Molecular pathways of normal hematopoietic cell differentiation, as well as the mechanisms by which oncogenes disrupt this process, remain poorly understood. In normal hematopoietic progenitor cells, a program of specific gene expression orchestrates commitment and differentiation of mature cells to multiple different lineages. In acute leukemias, however, oncoproteins interfere with this genetic program, resulting in the unregulated proliferation of cells that no longer retain the capacity to differentiate normally. In acute myeloid leukemias (AMLs) many known myeloid oncoproteins can block the differentiation of normal progenitors cultured in vitro in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, neither the genetic events that underlie normal hematopoietic cell differentiation nor the mechanism through which leukemic oncoproteins interfere with the execution of the program of lineage differentiation are well understood.
A number of genes have been identified that are critically involved in various forms of leukemia. For example, the t(1;19) chromosomal translocation in humans results in the production of E2a-Pbx1, a chimeric oncoprotein containing the transactivation domains of E2a joined to the DNA-binding homeodomain protein Pbx1. E2a-Pbx1 causes T-cell and myeloid leukemia in mice, blocks differentiation in murine myeloid progenitor cells, and transforms fibroblasts. The mechanisms of differentiation arrest are likely accompanied by aberrant expression of tissue specific and developmentally regulated genes. This aberrant tissue specific gene expression is also found in the subset of pre-B cells containing the t(1:19) translocation in humans. The exact mechanism by which E2a-Pbx1 alters gene expression is unclear, but it appears to modulate transcription in cooperation with homoebox gene products. In human pre B-ALL, expression of E2a-Pbx1 correlates with the expression of EB-1, a tyrosine kinase signal transduction gene. Potentially, EB-1 overexpression could interfere with normal signal transduction pathways in proliferation and differentiation.
Primary cells and myeloid cell lines offer useful, but limited, models to approach questions regarding mechanisms of normal myeloid cell growth and differentiation, and how this process goes awry in leukemia. Although primary marrow progenitor cells demonstrate normal granulocytic and monocytic differentiation in IL-3 or GM-CSF, one is limited by the scarcity of cells, the difficulty in isolating homogeneous populations, and the inability to verify expression of non-transforming oncoproteins when using such progenitor cells to study the normal program of myeloid differentiation and the mechanisms by which oncogenes alter this program. Useful myeloid cell lines that demonstrate inducible differentiation in response to changes in cytokines include FDCPmixA4 (GM-CSF+granulocyte colony-stimulating factor [G-CSF]+macrophage colony-stimulating factor [M-CSF]), 32Dlc3 (G-CSF), M1-AML (IL-6), and FDB cells (GM-CSF), whereas those that respond to nonphysiologic stimuli include HL60 (high levels of retinoic acid [RA], 12-o-tetradecanoylphorbal 13-acetate [TPA], dimethyl sulfoxide [DMSO]), EML (GM-CSF and RA), MPRO (RA), NB4 (RA), and U937 cells (RA, TPA, DMSO, or vitamin D3). There are no lymphoid cell lines that demonstrate inducible differentiation. Although these lines supply an unlimited number of clonal cells, most are limited by the fact that they contain undefined genetic changes such that their differentiation is often incomplete, asynchronous, or accompanied by cell death. Because the myeloid cells are already blocked to differentiation in response to either IL-3 or GM-CSF, it is unclear whether induction by other extrinsic factors proceeds through normal differentiation pathways. Furthermore, oncoproteins whose action it is to block differentiation induced by IL-3 or GM-CSF cannot be assayed in these prearrested cell lines.
A murine cell line was derived by Hogg and coworkers using a c-Myb-ER fusion was described. However, clonal c-Myb-ER cell lines were not derived, nor were the cells assayed for their ability to score differentiation arrest by other oncoproteins (Hogg et al. (1997) Oncogene 15:2885-98). Thus, the cell line overcomes some of the problems associated with established cell lines; however, the cell line is not clonal. This results in problems with reproducibility and maintenance of expression constructs. It is unclear that such a system would serve as a good model for myelopoiesis or AML.
An optimal hematopoietic cell line model would (1) lack constitutive expression of interfering oncoproteins, (2) exhibit conditional and terminal differentiation in response to biologically relevant molecules such as interleukins and growth factors, and (3) be blocked in an undifferentiated state by common leukemic oncoproteins. None of the cell lines listed above meet all of these criteria.